Portable Systems for Metered Dispensing of Aggressive Liquids.

Precise metering in liquid dispensing applications often requires application-specific solutions due to incompatibilities of the sensor and actuator components with the dispensed liquids. Some reoccurring challenges are aggressive liquids that would damage the sensors or tubing, the need for sterile liquids while the pumps or sensors cannot be sterilized, or media that can clog the sensor channels. Two different dispensing systems are here presented where the dispensing flow rate or volume is indirectly measured through a coupled pressure change or airflow, thus avoiding contact between the sensor and liquid. The controlled pressure-driven dispensing (cPDD) system builds an overpressure in the liquid reservoir by pumping air and controls the opening of the liquid output valve based on the internal pressure development. The FlowCap system uses a liquid pump on the outlet, controlled by the measured inflow of air to the reservoir. Both systems are designed for compactness and portability and offer independent operation, as well as control and communication, over a wireless interface.

Hippocampal infection with HSV-1-derived vectors expressing an NMDAR1 antisense modifies behavior.

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Gene transfer of NMDAR1 subunit sequences to the rat CNS using herpes simplex virus vectors interfered with habituation.

1. The aim is to study some roles of the hippocampal NMDA receptor, by modifying the expression of the essential NR1 subunit, with temporal and spatial restrictions in the central nervous system (CNS) of the rat. 2. Due to their neurotropism and the size of inserts they can accomodate, herpes simplex virus type-1 (HSV-1) derived amplicon vectors were used to transfer sequences, either in sense (+) or antisense (-) orientations, of the NR1 subunit gene, or of the green fluorescent protein (GFP) gene, into the CNS. 3. Vector expression in cell lines was followed by GFP autofluorescence, immunofluorescence and western blot. 4. The vectors were inoculated into the dorsal hippocampus of adult male Wistar rats, which were evaluated for habituation to an open field, and then, for expression of the transgenes, by autofluorescence and western blot; the expression mainly happened in pyramidal cells of CA1. 5. The animals injected with vectors carrying the NR1(+) transgene showed habituation to the new environment, as also happened with rats injected with vectors carrying only the GFP transgene. 6. In contrast, animals injected with vectors carrying NR1(-) sequence, did not show habituation. This might be retrograde amnesia or disability to record the trace, suggesting that the NR1 subunit in the dorsal hippocampus, is involved in habituation to a new environment. 7. HSV-1 derived amplicon vectors appear to be useful tools to modify endogenous gene expression, at a defined period, in restricted regions of the CNS.

Role of the membrane form of human colony-stimulating factor-1 (CSF-1) in proliferation of multipotent hematopoietic FDCP-mix cells expressing human CSF-1 receptor.

Because IL-3-dependent multipotential FDCP-Mix cells expressing human colony-stimulating factor-1 (CSF-1) receptor did not proliferate in response to soluble CSF-1, we investigated whether their proliferation would be induced in co-culture with adherent cells expressing the membrane form of CSF-1 (MemCSF-1). FDCP-Mix cells with high CSF-1R expression (NAF21 cells) were placed on stromal MS-5 cells or STO fibroblasts expressing MemCSF-1 (2M-1 cells and STO-M2 cells, respectively), in absence of IL-3. NAF21 cells bound significantly to 2M-1 cells as compared to control FDCP-Mix cells. Adhesion of NAF21 cells was inhibited by anti-huCSF-1 antibodies, as well as anti-huCSF-1R antibodies. Interestingly, NAF21 cells proliferated on both 2M-1 and STO-M2 cells but with very different kinetics. Moreover, NAF21 cell proliferation was also supported by glutaraldehyde-fixed 2M-1 cells or highly concentrated MS-5 cell culture supernatant, but not by CSF-1 coated on culture dishes. These results strongly suggest that MemCSF-1/CSF-1R interaction mediates a specific adhesion of NAF21 cells to stromal cells and allows stimulation of hematopoietic cells by stromal cell-derived factors expressed in a membrane-bound form or concentrated within the extracellular matrix. Thus, cytokine receptors deficient in mitogenic signalling may nevertheless have a regulatory role in hematopoietic progenitor cell proliferation by acting as adhesion molecules.

Expression and activity of caspases 1 and 3 in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.

Colony-stimulating factor-1 impairs both proliferation and differentiation signals of erythropoietin during the commitment of bipotential NFS-60 cell line to the monocytic lineage.

The interleukin-3 (IL-3) dependent cell line NFS-60 contains bipotential progenitors that exhibit both erythroid and myelomonocytic potentials. In order to study their commitment to the monocytic lineage, NFS-60 cells were retrovirally transduced with mouse c-fms cDNA, which encodes the colony-stimulating factor-1 receptor (CSF-1R), resulting in the N-Fms cell line. N-Fms cells proliferated in response to CSF-1 with a growth rate similar to that obtained in response to IL-3 and progressively differentiated from myeloid blasts to monocytic cells within 3 days of culture. When maintained in IL-3, about 3% of N-Fms cells formed large hemoglobinized colonies in semisolid cultures supplemented with erythropoietin (EPO). However, this property was lost after a 24-hour cultivation in the presence of CSF-1 or, interestingly, both CSF-1 and IL-3. This loss of response to EPO was reverted following a brief passage (24 hours) in IL-3, but the rescued colonies did not undergo terminal erythrocytic differentiation. Furthermore, CSF-1 also affected proliferative response to EPO of N-Fms cells constitutively expressing EPO receptors. Our data strongly suggest that CSF-1 can suppress erythroid potential in bipotential N-Fms cells by altering proliferative and differentiation signal of EPO.

A novel growth-factor-dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential.

Constitutive expression of human colony-stimulating factor-1 receptor (CSF-1R) confers long-lasting CSF-1-dependent proliferation to mouse myeloid cell lines. We developed mice transgenic for human CSF-1R because mouse CSF-1 cannot activate human CSF-1R. Then bone marrow cells from transgenic mice were plated onto MS-5 stromal cells expressing the membrane form of human CSF-1 (2M-1 cells) in order to combine the hematopoietic supporting properties of stromal cells and the proliferative effects of CSF-1. Thus, we were able to derive a hematopoietic cell line, called 47.10, that grew indefinitely under these conditions, whereas no cell line could be developed from nontransgenic mice. Proliferation of 47.10 cells is severely affected by neutralizing anti-CSF-1R monoclonal antibodies. Morphologic and cytofluorometry analysis established that most 47.10 cells are immature myelomonocytic cells. Consistent with this phenotype, the myeloid transcription factor PU.1, but not the erythroid transcription factor GATA-1, is expressed in 47.10 cells. A few 47.10 cells (3-5%) do not express lineage specific markers; they differentiate spontaneously to lineage-positive cells after replating on 2M-1 cells. In agar cultures, 47.10 cells form 7- and 14-day colonies in response to a cocktail of granulocyte/macrophage colony-stimulating factor (2.5 ng/mL), interleukin-3 (1 ng/mL), and mouse CSF-1 (10 ng/mL). Under these conditions, about 0.5% of 47.10 cells formed large 14-day colonies (>1 mm) composed of mature monocytes and granulocytes, reflecting the presence of progenitors endowed with high proliferative potential (HPP-47.10 cells). In conclusion, we have characterized a novel continuous myeloid cell line presenting a hierarchical structure similar to that of the bone marrow progenitor cell compartment.